This paper reports experimental research undertaken to study the effect of variation of inter-electrode distance on the Surface Electromyogram (SEMG). The experiments were conducted on ten healthy subjects and they performed isometric contraction of their biceps of the right arm at 20, 50 and 80% of their maximal voluntary contraction (MVC). SEMG was recorded using surface electrodes with the distance between the two active electrodes was maintained at 18 and 36 mm while the distant electrode position was fixed for all the experiments. It has been observed that at low levels of muscle contraction there was no significant variation due to the change in the distance between the electrodes while at 50 and 80% of MVC there was a significant change in the amplitude of the SEMG measured by the continuous RMS of the signal. The study has confirmed the well known belief that there is an increase in the RMS of SEMG with muscle contraction but the clearly demonstrates that comparison should only be done if the distance between the electrodes is kept constant.